The androgen receptor interacts with GATA3 to transcriptionally regulate a luminal epithelial cell phenotype in breast cancer

Background The androgen receptor (AR) is a tumor suppressor in estrogen receptor (ER) positive breast cancer, a role sustained in some ER negative breast cancers. Key factors dictating AR genomic activity in a breast context are largely unknown. Herein, we employ an unbiased chromatin immunoprecipitation-based proteomic technique to identify endogenous AR interacting co-regulatory proteins in ER positive and negative models of breast cancer to gain new insight into mechanisms of AR signaling in this disease. Results The DNA-binding factor GATA3 is identified and validated as a novel AR interacting protein in breast cancer cells irrespective of ER status. AR activation by the natural ligand 5α-dihydrotestosterone (DHT) increases nuclear AR-GATA3 interactions, resulting in AR-dependent enrichment of GATA3 chromatin binding at a sub-set of genomic loci. Silencing GATA3 reduces but does not prevent AR DNA binding and transactivation of genes associated with AR/GATA3 co-occupied loci, indicating a co-regulatory role for GATA3 in AR signaling. DHT-induced AR/GATA3 binding coincides with upregulation of luminal differentiation genes, including EHF and KDM4B, established master regulators of a breast epithelial cell lineage. These findings are validated in a patient-derived xenograft model of breast cancer. Interaction between AR and GATA3 is also associated with AR-mediated growth inhibition in ER positive and ER negative breast cancer. Conclusions AR and GATA3 interact to transcriptionally regulate luminal epithelial cell differentiation in breast cancer regardless of ER status. This interaction facilitates the tumor suppressor function of AR and mechanistically explains why AR expression is associated with less proliferative, more differentiated breast tumors and better overall survival in breast cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-023-03161-y.


Fig. S2 :
Fig. S2: Activation of AR induces GATA3 chromatin binding at AR/GATA3 co-occupied loci in ZR-75-1 breast cancer cells.Replicate data for GATA3 ChIP-seq in vitro experiments in T-47D (A) and ZR-75-1 (B) cells associated with Fig 2. Venn diagrams show the overlap of three independent experiments representing consecutive passages of cells treated with vehicle (Veh; EtOH), estradiol (E2; 10 nM), DHT (10 nM) or E2 + DHT (10 nM each).Peaks present in at least 2 of 3 replicates were used to generate a consensus cistrome, indicated below the Venn diagrams, for further comparative analyses.(C) Volcano plot reporting the FDR adjusted p-value and the log2FC of GATA3 chromatin binding events in ZR-75-1 (ZR)

Fig. S3 :
Fig. S3: GATA3 chromatin occupancy is altered by hormone treatment of ER-positive breast cancer cells.(A) Volcano plot reporting the FDR adjusted p-value and the log2FC of GATA3 chromatin binding events in ZR-75-1 breast cancer cells treated in vitro with E2 vs Veh.Threshold is set as in Figure 2. (B) Venn diagram showing the overlap of significantly enriched (FDR < 0.05) GATA3 binding sites with E2 or DHT in ZR-75-1 cells.(C) Transcription factor binding score at GATA3 DHT-induced loci (FDR < 0.05) or E2-induced loci in T-47D cells or (D) ZR-75-1 cells.Circles represent public data from breast cancer cell lines/tissue while triangles are derived from prostate cancer cell data, with color denoting cell line.(E)Volcano plot reporting the FDR adjusted p-value and the log2FC of GATA3 chromatin binding events with simultaneous hormone treatment (E2+DHT) compared to DHT alone in ZR-75-1.For visualization of differential binding patterns, the threshold for gain or loss in volcano plots

Fig. S4 :
Fig. S4: Activation of AR induces changes in GATA3, AR and H3K27ac chromatin binding in ER-negative breast cancer cells.Replicate data for GATA3 ChIP-seq, AR ChIPseq and H3K27ac ChIP-seq in MDA-MB-453 (A) cells and MFM-223 (B) cells.Venn diagrams show the overlap of three independent experiments representing consecutive passages of

Fig. S5 :
Fig. S5: Activation of AR induces binding at shared AR and GATA3 loci in ER-breast cancer cell lines.Venn diagram showing overlap of GATA3 and AR binding sites significantly

Fig. S6 :
Fig. S6: GATA3 promotes, but is not essential for, expression of shared AR and GATA3 target genes.(A) Representative immunofluorescence images showing AR silencing in MDA-MB-453 cells.Scale bar = 30 µm.(B) Western blot confirming AR silencing in T-47D and MDA-MB-453 cells.(C) mRNA expression of DHT-regulated AR target genes following AR silencing by two different siRNAs in T-47D cells (upper panel) and MDA-MB-453 cells, treated in vitro with Veh (EtOH) or DHT related to Fig 5B.Data was analyzed by a two-way ANOVA.Posthoc analyses were performed using Tukey's multiple comparisons test, where SEC14L2, P < 0.0001 for Veh versus DHT in siControl only; ZBTB16, P < 0.0001 and P < 0.01 for Veh

Fig. S7 :
Fig. S7: AR agonist-induced shared AR and GATA3 target genes are associated with development and differentiation of mammary epithelium.(A) RNA-seq heatmap of differentially expressed (FDR < 0.05) mammary lineage marker genes with DHT-induced shared AR and GATA3 binding in ZR-75-1 and (B) MFM-223 cells.Luminal marker genes are denoted by pink squares (y-axis) and basal marker genes are denoted by blue squares.Gene expression is represented as the log counts per million (logCPM) to observe patterns of expression between genes (rows).Genes common to all ER+ (T-47D, ZR-75-1) and ER-(MDA-MB-453, MFM-223) cell lines are indicated in purple.(C) Example genome browser images showing averaged GATA3, AR and H3K27ac ChIP-seq signals at binding sites

Fig. S8 .
Fig. S8.GATA3 loss disrupts expression of shared luminal epithelial shared AR/GATA3 target genes.GATA3 ChIP-PCR at AR/GATA3 co-occupied loci and resultant mRNA expression of associated luminal lineage genes following AR silencing by two different siRNAs in T-47D (A) and MDA-MB-453 (B) cells treated in vitro with Veh (EtOH) or DHT.Data was analyzed by a two-way ANOVA.Post-hoc analyses were performed using Tukey's multiple comparisons test, where **** indicates P < 0.0001; *** indicates P < 0.001; * indicates P<0.05.AR ChIP-PCR at AR/GATA3 co-occupied loci and resultant mRNA expression of associated luminal lineage genes following GATA3 knockdown in T-47D (C) and MDA-MB-453 (D) cells treated in vitro with Veh (EtOH) or DHT.Data was analyzed by a two-way ANOVA.Post-hoc analyses were performed using Tukey's multiple comparisons test, where **** indicates P < 0.0001 and *** indicates P < 0.001.Data is shown as mean ± SEM of 3 biological replicates from consecutive passages of cells.(E) Survival analyses using the KM plotter mRNA database to stratify KDM4B expression in breast cancer (all cases).(F) Western blot for FOXA1, AR, GATA3 and ER in ER+ (T-47D, ZR-75-1) and ER-(MDA-MB-453, MFM-223) cycling cell lines grown in regular culture media.